title: Calmodulin-dependent binding to the NHE1 cytosolic tail mediates activation of the Na+/H+ exchanger by Ca2+ and endothelin.
abstract: The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton (H(+)) in exchange for one extracellular Na(+). The human protein contains a ∼500-amino acid membrane domain and a regulatory, ∼315-amino acid cytosolic domain. NHE1 is activated by a number of hormones including endothelin (ET) and by Ca(2+). The regulatory tail possesses an inhibitory calmodulin (CaM)-binding domain, and inhibition of NHE1 is relieved by binding of a Ca(2+)-CaM complex. We examined the dynamics of ET-1 and Ca(2+) regulation of binding to NHE1 in vivo. CFP was linked to the NHE1 protein cytoplasmic COOH terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted properly and retained NHE1 activity comparable to the wild-type protein. We examined the in vivo coupling of NHE1 to CaM by Förster resonance energy transfer using CaM linked to the fluorescent protein Venus. CaM interaction with NHE1 was dynamic. Removal of serum reduced CaM interaction with NHE1. Addition of the Ca(2+) ionophore ionomycin increased the interaction between CaM and NHE1. We expressed an ET receptor in AP-1 cells and also found a time-dependent association of NHE1 with CaM in vivo that was dependent on ET treatment. The results are the first demonstration of the in vivo association of NHE1 and CaM through ET-dependent signaling pathways.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Xiuju
authors.initials: X
authors.lastname: Li
authors.forename: Daniel
authors.initials: D
authors.lastname: Prins
authors.forename: Marek
authors.initials: M
authors.lastname: Michalak
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D003412:Cricetulus; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D016232:Endothelins / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D027682:Cation Transport Proteins; D016232:Endothelins; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D002118:Calcium
keywords: calmodulin; endothelin; pH regulation; sodium/hydrogen exchanger